Reactive Oxygen Species (ROS) include oxygen-centered free radicals such as
superoxide (O2•-), hydroxyl radical (HO•),
alkoxy radical (RO•) and peroxyl radical (ROO•)
as well as nonradical species such as singlet oxygen (1O2)
and hydrogen peroxide (H2O2). These radical and nonradical
ROS are formed in living organisms during the normal metabolic processes. ROS,
especially free radicals are chemically very reactive and can attack molecules
in cells or tissues. For example, ROS can react with lipids in cell membranes,
proteins in tissues or enzymes and bases in DNA (Denisov and
Afanasve, 2005). This oxidative damage is believed to be a primary
factor not only in various diseases but also in the normal process of aging
(Valko et al., 2007). Humans have evolved with
antioxidant systems to protect against free radicals, which include enzymatic
defense such as superoxide dismutase, catalase and glutathione peroxidase. Through
these enzymes, superoxide and hydrogen peroxides are metabolized and therefore
production of detrimental hydroxyl radical is prevented. Even though this endogenous
defense system is provided, under some physiopathological situations such as
air pollutants, UV radiation and inflammation, ROS is produced in excess. In
order to diminish the cumulative effects of oxidative damage, exogenous antioxidants
are needed. Vitamins (A, C and E) and flavonoids from plant sources are antioxidants
in diet (Pietta, 2000). Antioxidants are applied in the
food industry as well as in the cosmetic industry as the functional ingredient
to prevent oxidative damages (Elzaawely and Tawata, 2012;
Gajula et al., 2009; Ham
et al., 2010). Since application of antioxidants become broad in
various areas, it is necessary to develop different type of novel antioxidative
agents. Especially, natural antioxidants from plant sources are more favorable
in the industry due to their environmentally friendly properties (Adisa
et al., 2011; Chanda et al., 2011;
Hajimahmoodi et al., 2008; Oboh
and Ademosun, 2006).

We are continuously conducting phytochemical studies on plants growing in Jeju,
the largest island located at the southernmost part in Korea (Kim
et al., 2010a, b; Kim
et al., 2011; Ko et al., 2011). In
the course of our investigation for the biologically active natural products,
we observed antioxidative activities in the ethanol extract prepared from the
stems of Cleyera japonica, which led us to identify the active constituents.

C. japonica Thunb. (Theaceae family) is an evergreen tree of height
up to 10 m distributed over Korea, Japan and Taiwan. This tree has usually been
used as lumber for household furniture in Korea. The acetone extract from the
leaves of C. japonica var. morii collected in Taiwan
showed a strong free radical scavenging activities (Hou
et al., 2003). However, no chemical constituents have been presented
responsible for the activities. We herein described the antioxidative constituents
from the ethanol extract of C. japonica stems and their scavenging activities
against DPPH, hydroxyl and superoxide anion radicals.

MATERIALS AND METHODS

Reagent and equipment: All solvents used in this experiment were of analytical grade. 1H (400 MHz) and 13C (100 MHz) NMR spectra were recorded on a JEOL (JNM-ECX 400) instrument with chemical shift data reported in ppm relative to the solvent used. JES-FA200 (JEOL) Electron Spin Resonance (ESR) spectrometer was used for the radical scavenge tests. Merck silica gel (0.063-0.2 mm) was used for normal phase column chromatography. Silica gel 60 F254 coated on aluminum plates by Merck were used for Thin Layer Chromatography (TLC). Gel Filtration Chromatography (GFC) was performed using Sephadex LH-20 (25100 μm) from Fluka. DPPH (2,2-diphenyl-1-picrylhydrazyl) was purchased from Aldrich. Xanthine, xanthine oxidase and DMPO (5,5-dimethyl-1-pyrroline N-oxide) were purchased from Sigma.

Plant material: The stems of C. japonica Thunb. were collected from the Halla Botanical Garden, Jeju Island in Korea. A voucher specimen (No. 124) was prepared and deposited at the laboratory of Natural Product Chemistry, Department of Chemistry, Jeju National University.

DPPH radical scavenging activity: Sample solutions (20 μL) of different concentrations (100, 50, 25, 12.5, 6.25 and 3.125 μg mL-1 in DMSO) were added to a 0.2 mM DPPH ethanol solution (180 μL) and allowed to react at room temperature. The absorbance values were measured after 10 min at 515 nm with a UV/Vis spectrophotometer. The DPPH radical scavenging activities of samples were calculated according to the formula:

where, Abssample is the absorbance of the experimental sample, Absblank is the absorbance of the blank, Abscontrol is the absorbance of the control. Ascorbic acid (vitamin C) was used as a positive control. Each treatment was replicated thrice.

DPPH radical scavenging activity with ESR: Test solution was prepared
by mixing 10 μL of sample and 90 μL of DPPH (0.2 mM) in a methanol
solution. After mixing vigorously for 10 sec, the solution was then transferred
into a 100 μL Teflon capillary tube fitted into the cavity of the ESR spectrometer.
The ESR spectrum was recorded 2 min after test solution preparation. ESR spectrometer
parameters were set as follows: magnetic field of 337 mT, power of 1.00 mW,
frequency of 9.4375 GHz, modulation amplitude of 0.8 mT, gain of 500, scan time
of 0.5 min, scan width of 10 mT and time constant of 0.03 sec at room temperature
(Kim et al., 2010c). Ascorbic acid was used as
a positive control and each treatment was replicated thrice.

Hydroxyl radical scavenging activity: Hydroxyl radicals generated by
the Fenton reaction (H2O2 plus FeSO4) were
reacted with a radical spin trap, DMPO. The resulting DMPO-OH radicals were
detected by using an ESR spectrometer. Briefly, ESR signaling was recorded after
2.5 min of test solution preparation by mixing of sample (10 μL) with 0.3
M DMPO (30 μL), 10 mM FeSO4 (30 μL) and 10 mM H2O2
(30 μL). ESR spectrometer parameters were set as follows: magnetic field
of 337 mT, power of 1.00 mW, frequency of 9.4354 GHz, modulation amplitude of
0.2 mT, gain of 200, scan time of 0.5 min, scan width of 10 mT and time constant
of 0.03 sec at room temperature (Kim et al., 2010c).
Ascorbic acid was used as a positive control and each treatment was replicated
thrice.

Superoxide anion radical scavenging activity: Superoxide anion radicals
produced by a xanthine/xanthine oxidase system were reacted with the spin trap
agent, DMPO. The generated DMPO-OOH radicals were detected using ESR spectrometry.
Briefly, ESR signaling was recorded 5 min after of test solution preparation
by mixing of sample (10 μL) with 1.5 M DMPO (30 μL), 5 mM xanthine
(30 μL) and 0.25 U mL-1xanthine oxidase (30 μL). The parameters
of the ESR spectrometer were: magnetic field of 337 mT, power of 5.00 mW, frequency
of 9.4374 GHz, modulation amplitude of 0.2 mT, gain of 700, scan time of 0.5
min, scan width of 10 mT, time constant of 0.03 sec and a temperature of 25°C
(Kim et al., 2010c). The ascorbic acid was used
as a positive control and each treatment was replicated thrice.

Statistical analysis: Means±SEM of the data were calculated;
statistical analysis of the results was performed by Student's t-test for independent
samples. Values of p<0.05 were considered significant.

RESULTS AND DISCUSSION

In the course of preliminary screenings, the ethanol extract of C. japonica
stems was found to have significant radical scavenging activities. These antioxidative
activities were measured against DPPH and hydroxyl radicals by using Electron
Spin Resonance (ESR) spectrometry. ESR spectroscopy, whose signal intensities
depend on the concentrations of free radicals, is a sensitive and specific method
for the detection of radical species in chemical and biological systems. Direct
measurement of radical species with ESR in the reaction mixture has advantages
over traditional spectrophotometric method leading to high reliability and precision.
This technique has been successfully applied for systematic studies on the evaluation
of antioxidant capacity of natural extracts (Jiang et
al., 2010). Upon the DPPH radical inhibition activities, the ethanol
extract exhibited SC50 13.6 μg mL-1 which was comparable
to ascorbic acid (SC50 4.6 μg mL-1; positive control).
In addition, the extract was comparable (SC50 442.5 μg mL-1)
to ascorbic acid (SC50 153.6 μg mL-1) in the hydroxyl
radical inhibition tests.

As the ethanol extract exhibited considerable free radical inhibition properties,
a phytochemical study was conducted in order to identify the active constituents.
The extract was partitioned successively into n-hexane, ethyl acetate (EtOAc),
n-butanol and water soluble fractions. The EtOAc fraction was chosen and subjected
to repeated column chromatography over silica gel and Sephadex LH-20.

The antioxidant properties for compounds (1-7) were examined in radical inhibition
assays. DPPH (2,2-diphenyl-1-picrylhydrazyl) scavenging activities can be assayed
by a spectrophotometer and it forms a stable radical species with a strong absorption
at 515 nm bearing purple color; its degradation by test sample can be monitored
by disappearance of absorption. Using the DPPH radical inhibition assay, all
isolates 1-7 showed more potent activities than the positive control ascorbic
acid (SC50 44.9 μM) (Table 1). Proanthocyanidin
A-1 (7) displayed the most potent activity. The disappearance of DPPH radical
species can also be monitored by an ESR spectrum.

As shown in Table 2, this assay also showed that compound
7 was the most potent inhibitor with a SC50 of 9.4 μM, indicating
better activity than ascorbic acid (SC50 23.3 μM). The ESR spectrum
for compound 7 is shown in Fig. 2.

As hydroxyl radical is the most reactive chemical species among ROS, this species
produces the most deleterious effect on cells in living organisms (Denisov
and Afanasve, 2005). In the scavenging activity assays, hydroxyl radical
was generated by the reaction of hydrogen peroxide and Fe2+ ion known
as the Fenton reaction. The hydroxyl radical inhibition was verified using ESR
by monitoring the DMPO-OH radical peak. In this experiment, the reactive hydroxyl
radical was trapped by a nitrogen N-oxide, DMPO to yield a relatively stable
radical DMPO-OH, which can be detected by ESR. Using this hydroxyl radical inhibition
assay, compound 7 exhibited more potent activity (SC50 301.6 μM)
than ascorbic acid (SC50 859.7 μM) (Table 3).
Figure 2 indicated that hydroxyl radical was scavenged by
compound 7 in a dose-dependent manner. The other compounds appeared to have
relatively lower activities.

Superoxide anion is a ROS generated during the respiratory metabolic process
in mitochondria. Even though this radical anion is not as reactive as hydroxyl
radicals, it can initiate a cascade of ROS.

In conclusion, phytochemical investigation of the stems of C. japonica led to the isolation of seven compounds. We demonstrated that compounds 1-7 possessed relatively potent inhibition activities against DPPH, hydroxyl and superoxide anion radicals. Natural antioxidants may be responsible for the protective effects against the risk of many physiological and pathological processes. Based on these results, it is suggested that the extract of C. japonica stems containing potent antioxidative constituents could have potential in many industrial applications.

ACKNOWLEDGMENT

This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2011-0007254).